Molecular basis of the persistence of chloramphenicol resistance among Escherichia coli and Salmonella spp. from pigs, pork and humans in Thailand

This study aimed to investigate the potential mechanisms associated with the persistence of chloramphenicol (CHP) resistance in Escherichia coli and Salmonella enterica isolated from pigs, pork, and humans in Thailand. The CHP-resistant E. coli (n = 106) and Salmonella (n = 57) isolates were tested for their CHP susceptibility in the presence and absence of phenylalanine arginine β-naphthylamide (PAβN). The potential co-selection of CHP resistance was investigated through conjugation experiments. Whole genome sequencing (WGS) was performed to analyze the E. coli (E329, E333, and E290) and Salmonella (SA448, SA461, and SA515) isolates with high CHP MIC (32–256 μg/mL) and predominant plasmid replicon types. The presence of PAβN significantly reduced the CHP MICs (≥4-fold) in most E. coli (67.9%) and Salmonella (64.9%). Ampicillin, tetracycline, and streptomycin co-selected for CHP-resistant Salmonella and E. coli-transconjugants carrying cmlA. IncF plasmids were mostly detected in cmlA carrying Salmonella (IncFIIAs) and E. coli (IncFIB and IncF) transconjugants. The WGS analysis revealed that class1 integrons with cmlA1 gene cassette flanked by IS26 and TnAs1 were located on IncX1 plasmid, IncFIA(HI1)/HI1B plasmids and IncFII/FIB plasmids. IncFIA(HI1)/HI1B/Q1in SA448 contained catA flanked by IS1B and TnAs3. In conclusion, cross resistance through proton motive force-dependent mechanisms and co-selection by other antimicrobial agents involved the persistence of CHP-resistance in E. coli in this collection. Dissemination of CHP-resistance genes was potentially facilitated by mobilization via mobile genetic elements.


Introduction
One of the primary drivers to the emergence and spread of antimicrobial resistance (AMR), a serious global public health threat, is the indiscriminate use of antimicrobials (AMU) [1].AMR develops more rapidly through the inappropriate and excessive use of antimicrobial agents and therefore, reducing or ceasing the use of antimicrobials is expected to lessen the likelihood of AMR bacteria development and distribution [2].In other words, AMR would have vanished if the AMU had been removed.
In contrast, bacteria resistant to restricted antimicrobial drugs have been consistently isolated.For example, the use of chloramphenicol in food animals has been banned in many countries e.g., the US, Canada, Australia, Japan and China since 1994 due to being a cause of aplastic anemia [3].The antibiotic has been outlawed in Thailand since 1998 [4]; nonetheless, chloramphenicol-resistant bacteria are continuously isolated from food animals and meat e.g., pig [5], poultry [6], pork and chicken [7,8].It was suggested to be the result of co-selection or cross-resistance brought on by other antimicrobials [9][10][11].
Chloramphenicol is a broad-spectrum antibiotic in the amphenicol group, which inhibits bacterial protein synthesis by binding to 50s ribosomal subunit [12,13].One of the most common mechanisms of chloramphenicol resistance is its enzymatic inactivation, particularly by cat-encoded acetyltransferases [14].Another possible resistance mechanisms include the expression of efflux pumps, which frequently function as multidrug extrusion transporters, reduced outer membrane permeability, and target site mutation or alteration.The cml and floR genes encode specific exporters of chloramphenicol, while the AcrAB-TolC multidrug efflux system can also export chloramphenicol, but to a lesser degree [15].Several chloramphenicol resistance genes (e.g., catA, catB, cmlA and floR) were found to be located on either transposon (e.g., Tn9 and Tn2424) and plasmids.In certain plasmids (e.g., IncX plasmid), chloramphenicol resistance genes were preserved, rather than in others [16].These genes were found co-located on the same conjugative plasmid with the other AMR genes such as tet, aadA and sul, conferring multidrug resistance phenotype [17].Despite being banned in food animals, chloramphenicol is used topically to treat eye infections in humans.It has antibiofilm activity, hypothetical low impact on ocular microbiota and narrow resistance rate [18].There might be the resuscitation of an old antimicrobial medication such as chloramphenicol in the situation that newer medications are not readily available due to the AMR issue.Therefore, investigating the mechanisms behind the persistence of chloramphenicol resistance in the absence of the antibiotic is worthwhile.
To date, Whole Genome Sequencing (WGS) has become an indispensable technique for AMR research and control, with the potential for the discovery of novel antibiotics, identification of AMR in clinical samples, AMR epidemiological surveillance, and tracking of AMR emergence [19].Information generated by WGS is greatly beneficial for comprehending the origins and transmission of AMR as well as the foundation of AMR mechanisms.This study aimed to determine possible mechanisms associated with the persistence of chloramphenicol resistance in Escherichia coli and Salmonella enterica isolated from pigs, pork, and humans.WGS was applied to allow genome-wide analysis of CHP-resistant bacteria.

Bacterial isolates and antimicrobial susceptibilities
The E. coli (n = 106) and Salmonella (n = 57) isolates resistant to chloramphenicol (MIC� 32 μg/ml) were obtained from our previous AMR monitoring projects during 2007-2008.The E. coli isolates were obtained from fecal content on rectal swabs of clinically healthy pigs at slaughterhouses.The rectal swab samples were immediately taken from pigs after stunning and bleeding but before the scalding process.E. coli were isolated and biochemically confirmed [20,21] and a single colony of E. coli of each positive sample was collected.Minimum inhibitory concentration (MIC) was determined using agar dilution technique [22].The isolates exhibited resistance to ampicillin (AMP, 81.2%), chloramphenicol (CHP,100%), ciprofloxacin (CIP, 43.6%), gentamicin (GEN, 47%), streptomycin (STR, 57.3%), sulfamethoxazole (SMX, 67.5%), tetracycline (TET, 98.3%) and trimethoprim (TMP, 91.5%).All the pigs were raised in closed house system in large-scale commercial farming operations with between 11,000 and 13,000 pigs.According to the information provided by farm veterinarians, the antimicrobials routinely used were amoxicillin, chlortetracycline, tylosin, tiamulin, and fosfomycin.

Determination of efflux system inhibitor effects on chloramphenicol susceptibility
The MIC value of CHP (Sigma-Aldrich, Saint Louis, MO) was determined in the presence and absence of 25 μg/mL phenylalanine arginine β-naphthylamide (PAβN, Sigma-Aldrich) using two-fold agar dilution method [22].The concentrations of CHP ranged from 1 μg/mL to 1,024 μg/mL.A 4-fold or more change in the chloramphenicol MIC value following the addition of PaβN was defined as significant.Experiments were repeated on two separate occasions.

PCR based replicon typing (PBRT)
DNA templates were prepared using whole cell boiling method [24].All PCR amplification was performed using Top Taq Master Mix kit (QIAGEN, Hilden, Germany) according to the manufacturer's instruction.All primers used in this study are listed in Table 1.Eighteen replicons including HI1, HI2, I1, X, L/M, N, FIA, FIB, W, Y, P, FIC, A/C, T, FIIAs, F repB , K and B/ O were PCR amplified using the following thermocycles: one cycle of denaturation at 94˚C for 5 min; followed by 30 cycles of denaturation at 94˚C for 1 min, annealing at 60˚C for 30 s, and extension at 72˚C for 1 min; and a final extension at 72˚C for 5 min.The exception was for F simplex PCR using F repB primers which employed an annealing temperature of 52˚C.

Conjugation experiment
Biparental mating was performed to determine co-selection of CHP resistance by other antibiotics.All the CHP-resistant E. coli (n = 106) and Salmonella (n = 57) isolates served as donors.Salmonella Enteritidis SE12rif R (CHP MIC = 4 μg/mL) [27] and E. coli MG1655rif R (CHP MIC = 4 μg/mL) [28] were used as recipients for the E. coli and Salmonella donors, respectively.Neither E. coli MG1655rif R nor Salmonella SE12rif R carry any of the 18 replicons tested.Transconjugants were selected on Luria Bertani agar containing rifampicin (32 μg/mL) and

Statistical analysis
The descriptive statistic including percentage was analyzed by excel program.The chi-squared test and z-test using Bonferroni method with SPSS version 22.0 program was used to compare the effect of PAβN on MIC values of chloramphenicol.A p-value of <0.05 was considered statistically significant.

Effect of efflux pump inhibitor to chloramphenicol MIC
Effects of PAβN on CHP MICs were determined in all E. coli (n = 106) and Salmonella (n = 57) (

Genomic characteristics of CHP-resistant E. coli and Salmonella
The quality of genome assembly of selected E. coli and Salmonella is shown in Table 5.The genome size and GC content of E290 (serotype O8:H16), E329 (serotype O37:H34), and E333 For Salmonella, genome size and GC content in Salmonella Weltevreden SA448, Salmonella Rissen SA461 and Salmonella Rissen SA515 were 5,419,175 bp, 51.86%; 5,127,858 bp, 52.05%, and 5,214,816 bp, 52.06%, respectively (Table 5).SA448 and SA461 comprised 4 contigs, including one for chromosome and 3 for plasmids.There were 11 contigs in SA515 including 3 for plasmids and 8 for chromosomes.
Structural comparison of plasmid carrying cmlA1, catA and floR.IncFIA(HI1)/HI1B plasmid from E329 and E333 and IncFII/FIB plasmid from SA461 and SA515 shared similar structure and sequences (Fig 2).AMR genes and ISs in two regions located upstream and  downstream of IncFIA(HI1)/HI1B plasmids are distributed in IncFII/FIB and IncX1 plasmid.

Discussion
It is becoming more well recognized that bacteria can continue to develop resistance even when antibiotics are banned, of which CHP resistance is among the most well-known examples of this phenomena.Our findings demonstrated that the mechanisms involved in the persistence of CHP resistance in E. coli and Salmonella, including cross-resistance mediated by the expression of multidrug efflux systems using proton motif force and co-selection of R plasmids by other antimicrobial agents.All E. coli and Salmonella in this study were resistant to CHP, of which most E. coli (67.9%) and Salmonella (56.1%) exhibited �4-fold CHP MIC decrease when PAβN is present, indicating the involvement of the RND multidrug efflux systems in their CHP resistance phenotype.Hence, any antimicrobial agent that activates the expression of a multidrug efflux pump (s) may promote cross resistance to CHP.At the same time, most CHP-resistant E. coli and Salmonella with <4-fold reduction of CHP MICs (0 to � 2 folds) in the presence of PAβN, carried cmlA encoding a proton motif force dependent multidrug efflux pump that belongs to Major Facilitator superfamily.This implies that the CmlA multidrug efflux pump confers a low-level resistance to CHP.
A plasmid borne-or chromosomally encodedcat gene encodes chloramphenicol acetyltransferase (CAT) enzyme.The genes were common in the CHP-resistant E. coli and Salmonella (41% and 51%, respectively), in agreement with previous studies [48,49].The contribution of RND efflux pumps in CHP resistance was limited in the CHP-resistant Salmonella carrying catA and catB (<4-fold CHP MIC reduction).In contrast, the presence of PAβN reduced CHP MIC �4 fold in E. coli carrying catA and catB, indicating the accumulative effects of enzymatic and non-enzymatic mechanisms on CHP resistance.
In conjugation experiment, AMP, TET and STR selective pressure co-selected for CHP resistance in E. coli or Salmonella transconjugants, similar to previous studies conducted in E. coli isolates from humans and food animals from United States [50].Almost all CHP-resistant E. coli or Salmonella transconjugants carried cmlA, while one CHP-resistant Salmonella transconjugants contained cat, indicating the localization of cmlA and cat on transferable R plasmids and in agreement with previous studies [51,52].However, the CHP MIC among Salmonella and E. coli transconjugants increased � 4 folds (from 4 to 32 and 4 to 128 folds, respectively) and was inconsistent with the limited contribution of cmlA suggested by the PAβN experiment.It was possible that the contribution of cmlA to CHP resistance level was more clearly observed in in vitro settings where the recipients with low CHP MIC were used (CHP MIC of 4 μg/ml for both E. coli MG1655rif r and Salmonella Enteritidis SE12 rif r ).
In the PBRT experiment, no plasmids were detected in some cmlA carrying Salmonella transconjugants (E292T_AMP, E294T_TET and E329T_STR), in agreement with their corresponded donors.The possibility exists that the gene was located on plasmids that were not part of the PBRT scheme used in this study.It was previously suggested that PBRT may be unable to detect some replicons on the large multiple-replicon plasmids due to mutation through transpositional alterations and unknown existence of the new replicons [53].It highlighted the necessity of expanding the PBRT to enable rapid plasmid screening.IncF plasmids were predominantly found in cmlA carrying transconjugants, in agreement with a previous study [52].This was not surprising because IncF plasmids are prevalent in Enterobacterales.In addition to IncF plasmids, IncHI1 and IncA/C plasmids were horizontally transferred (E331T_AMP, E333T_AMP, SA448T_TET, SA448T_STR, SA515T_TET, SA515T_STR) in agreement with a previous study conducted in Salmonella clinical isolates [52,54].Nevertheless, it remained unclear which plasmid carried cmlA and this could be a subject of future study.
According to WGS analysis, cmlA was identified on class1 integrons with dfrA12-aadA2-cmlA1-aadA1cassette array either located on IncX1 plasmid (in E290), in agreement with previous studies conducted in E. coli from human and livestock [55,56] or IncFIA(HI1)/HI1B (in E329 and E333).In SA461 and SA515 from pork, cmlA was found on IncFII/FIB plasmid carrying class 1 integrons carrying dfrA12-aadA2-cmlA1-aadA1 gene cassette array, in agreement with previous reports in Europe [57].The gene was additionally found located on class 1 integrons with estX-psp-aadA2-cmlA1-aadA1 cassette array in E. coli and Shigella from China and Taiwan [58,59].Besides, the cmlA gene was previously found on other transferable plasmids such as IncF, IncA/C, IncHI1 and IncR plasmids [52,60].As such, co-selection for cmlA1 conferring CHP resistance could occur when trimethoprim, streptomycin, and spectinomycin are used.The presence of cmlA on different class 1 integrons located on different plasmids could explain its widespread along the food chain.
As evidenced by a comparison of structural variations in WGS, several resistance genes resided on cmlA or catA-carrying plasmids.All plasmids (IncFIA(HI1)/HI1B, IncFII/FIB and IncX1 plasmids) carried class 1 integrons with dfrA12-aadA2-cmlA1-aadA1 cassette array.IncFII/FIB of SA461 and SA515 contained the same upstream and downstream genes as IncFIA(HI1)/HI1B of E329 and E333, but in a different order.In addition, the aph(3")-Ib, mcr3.1, tetM, aac(3)-IId and bla TEM-1 genes were found at the downstream of IncFIA(HI1)/ HI1B in E329 and E333.Notably, cmlA and bla TEM-1 were found together in all plasmids, similar to previous study [61].The class 1 integrons with dfrA12-aadA2-cmlA1-aadA1 cassette array were flanked by TnAs1/ Tn3 at upstream and IS6/ IS26 at downstream, in agreement with previous studies [62,63].TnAs1 and TnAs3 are members of the Tn21 family with Tn As3 more frequently linked to cmlA1 and intI1 [64].The mobilization of phenicol resistance genes is commonly mediated with transposons, particularly Tn21 [64], in agreement with the observation in this study.Flanking by insertion sequences facilitates the mobilization of many genes, whereas IS26 and other IS6 family members was previously shown to enhance the ability for replicon fusions, or the cointegration of donor and target replicons, contributing to the spread of the resistance determinants [65].Another evidence is that SA448 carried catA that was situated between IS1B and TnAs3 on IncFIA(HI1)/HI1B/Q1 plasmid, similar to a previous study [66].Besides, GCN5-related N-acetyltransferase (GNAT) encoding gene that is primarily implicated in resistance to CHP, aminoglycosides and streptogramins was inserted between catA and TnAs3 immediately upstream to IS6/IS26 [67].GNAT and a global regulator, clp, were linked and co-transcribed for CHP detoxification leading to CHP resistance.Taken together, these findings are evidence of how the co-selection of CHP resistance is mediated by other antibiotics.
According to the structural comparison, the nearly all sequences of the IncFIA(HI1)/HI1B/ Q1 plasmid of SA448 and the reference plasmid (accession no.CP022495.1)were identical with the exception of two horizontal gene transfer regions (HGT region) containing IS1B-catA-GNAT-TnAs3-IS26-IS6 and IS26-aph(6)-ld-aph(3")-lb-sul2-IS26.The CP022495.1 reference plasmid originated from Salmonella Derby; however, further details regarding origins and locations were absent.Regardless, the findings demonstrated the dynamics of AMR gene mobilization through R plasmids, contributing to AMR dissemination.
CHP is a substrate for AcrAB-TolC multidrug efflux pump.In this study, SA448, SA461 and SA515 carried point mutations in chromosomally encoded acrB, leading to amino L40P and F28L acid substitution of AcrB.The relation of the amino acid substitution to any specific AMR was not predicted due to a lack of the mutation available in the ResFinder database.The mutations suggested that the CHP resistance phenotype in the isolates was not attributed to the expression of this system.
There are limitations to this study that should be noted.Only PaβN that primarily reduces the efflux effect of RND efflux pumps was used, therefore, the effects of efflux pumps in different families were disregarded.Although WGS produces a massive amount of data, a lot of it could be irrelevant or ambiguous.Despite advances in genomics, many resistance genes are still not identified and not comprised in databases available for AMR analysis.In addition, the limited number of CHP-resistant isolates from pigs were included.Increasing the number of isolates from other livestock would reveal more insights of the mechanisms contributing to the CHP resistance persistence.
In conclusion, the results unveiled cross resistance by multidrug efflux system and co-selection of R plasmids by other antimicrobial drugs as key mechanisms, contributing to the persistence of CHP resistance in E. coli and Salmonella in this study.Reduction and optimization of antimicrobial consumption is necessary to combat AMR.It is evident that restricting the use of a single antimicrobial agent is insufficient to address the issue.In addition to the prohibition on antibiotic usage, several policies and initiatives that reduce the need for antibiotics and delay the spread of AMR are necessary e.g., farm biosecurity, infection control, vaccination program, prudent antimicrobial use etc. Veterinarians are advised to undertake antimicrobial stewardship to improve antimicrobial utilization and decrease the indiscriminate use of antibiotics.Laboratory-based AMR surveillance should be prioritized and conducted at phenotypic and genotypic level with coordination among sectors.

Fig 2 .Fig 3 .
Fig 2. Alignment of plasmids carrying cmlA1 of E. coli (n = 3) and Salmonella enterica (n = 2).(A) Whole plasmid sequences of IncFIA(HI1)/HI1B plasmid from E329 and E333, IncFII/FIB plasmid from SA461, SA515, and IncX1 plasmid from E290 were compared.Green arrows indicate the position and direction of genes.Blue vertical blocks indicate regions of shared similarity shaded according to BLASTn (dark blue for matches in the same direction and red for inverted matches).(B) A zoomed-in view (yellow dashed line-box) shows the area containing class1 integrons with cmlA1 gene cassette, which is flanked by Tn3-like element Tn3 or TnAs1 family transposase at upstream and IS6-like element of IS26 family transposase at downstream.(C) A zoomed-in view (gray dashed linebox) shows the position of bla TEM-1B and IS6-like element of IS26 family transposase that are located downstream of class1 integrons in IncFIA(HI1)/HI1B plasmid of E329 and E333 and IncX1 plasmid of E290 and upstream in IncFII/FIB plasmid from SA461 and SA515.https://doi.org/10.1371/journal.pone.0304250.g002